Stephen Douglas Johnston

University of Queensland | UQ · School of Agriculture and Food Sciences

Although monotremes diverged from the therian mammal lineage approximately 187 million years ago, they retain various plesiomorphic and/or reptilian‐like anatomical and physiological characteristics. This study examined the morphology of juvenile and adult female reproductive tracts across various stages of the presumptive oestrous cycle, collected...

The use of samples or scats to provide important ecological, genetic, disease and physiology details on free-range populations is gaining popularity as an alternative non-invasive methodology. Koala populations in SE Queensland and NSW have recently been listed as endangered and continue to face anthropomorphic and stochastic environmental impacts...

Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human...

It is possible that the reproductive strategy of the short-beaked echidna is related to seasonal changes in fat deposition and energy availability, regulated by seasonal changes in endocrine function. We predicted that circulating leptin levels would be directly proportional to adiposity during most of the year, but that a change in this relationsh...

The sex chromosomes of egg-laying mammals (monotremes), which lack the sex determining gene SRY, evolved independently to those of all therian mammals. Here we characterise the candidate monotreme sex determining gene, the Y-localised anti-Müllerian hormone gene (AMHY) and trace its expression during the period of sexual differentiation. Monotreme...

In this clinical era of intracytoplasmic sperm injection (ICSI), where a single spermatozoon is chosen for fertilization, the diagnostic functionality of the classical parameters typically associated with fertilization, such as sperm concentration, sperm motility, acrosome integrity, and mitochondria, is perhaps becoming less critical. In contrast,...

Objective To determine the relationship between teratozoospermia and sperm DNA fragmentation (SDF) in the human ejaculate. Methods This retrospective study included 100 normozoospermic men as a control cohort (abnormal forms >14%), 210 patients with a high level of abnormal forms (≤4%) and 65 patients presenting with a moderate level of abnormal f...

Chlamydiosis is one of the main causes of the progressive decline of koala populations in eastern Australia. While histologic, immunologic, and molecular studies have provided insights into the basic function of the koala immune system, the in situ immune cell signatures during chlamydial infection of the reproductive tract in koalas have not been...

Indigenous gut microbial communities (microbiota) play critical roles in health and may be especially important for the mother and fetus during pregnancy. Monotremes, such as the short‐beaked echidna, have evolved to lay and incubate an egg, which hatches in their pouch where the young feeds. Since both feces and eggs pass through the cloaca, the f...

Background While the kinetics of human sperm nuclear DNA fragmentation (SDF‐nDNA) following ejaculation have been described, the dynamics and relationships of mitochondrial DNA copy number per spermatozoon (mtDNAcn) and fragmentation (SDF‐mtDNA) remain unexplored. Objectives To compare post‐ejaculatory kinetics of mtDNAcn, SDF‐mtDNA and SDF‐nDNA,...

Simple Summary The koala has recently been listed as an endangered species in the northern part of its range where it faces significant challenges associated with habitat fragmentation, climate change, and disease. Fundamental to its conservation is a detailed knowledge of its reproductive system, particularly with respect to what represents normal...

The short‐beaked echidna is sexually monomorphic such that gender identificationwithout veterinary intervention is challenging. The aim of this study was to evaluateand compare the most optimal noninvasive genetic source by extracting echidnagenomic DNA (gDNA) from fecal scats, plucked hair, and quills to perform geneticsex testing using a range of...

Inbreeding threatens many species of conservation concern. Inbreeding decreases heterozygosity (increases homozygosity) and can drive up a population’s genetic load as deleterious mutations increase in frequency. Understanding how declining heterozygosity translates into declining fitness is of high importance for conservation practitioners. In thi...

Background While it is common to clinically evaluate sperm nuclear DNA fragmentation, less attention has been given to sperm mitochondrial DNA. Recently, a digital PCR assay has allowed accurate estimation of the proportion of fragmented mtDNA molecules and relative copy number. Objectives To determine the correlation of classical sperm parameters...

The prostate of the koala (Phascolarctos cinereus), and of marsupials more generally, is the primary contributor of seminal fluid, but comparatively little is known about its microanatomy and/or biochemistry. This study explored evidence of parenchymal segmentation of the koala prostate. The prostate of three sexually mature koalas, euthanised for...

Background Indigenous microbial communities (microbiota) play critical roles in health and may be especially important for mother and fetus during pregnancy. Having diverged from Eutherian mammals roughly 184 million years ago, monotremes, such as the short-beaked echidna, have evolved to lay and incubate an egg, which hatches in their pouch where...

This retrospective study assessed the biological intra-individual variability of the percentage of sperm with DNA damage (SDF) observed in subsequent ejaculates of the same individual. Variation in SDF was analyzed using the Mean Signed Difference (MSD) statistic based on 131 individuals, comprising 333 ejaculates. Either two, three or four ejacula...

The aim of this study was to investigate the relationship between DNase activity associated with bacterial contamination of incubated bovine frozen-thawed spermatozoa and elevated sperm DNA fragmentation. Electrophoresis analysis of plasmid PBR322 incubated for 30 min at 37 °C with the supernatant of the diluent of frozen-thawed centrifuged bovine...

In the echidna, after development in utero, the egg is laid in the pouch and incubated for 10 days. During this time, the fetuses develop an egg tooth and caruncle to help them hatch. Using rare and unprecedented access to limited echidna pre- and post-hatching tissues, the egg tooth and caruncle development were assessed by micro-CT, histology and...

Research question To examine the effect of a novel non-centrifugation method (IO-Lix) of sperm selection on sperm parameters and ICSI reproductive outcomes. Design A pilot study elevated the capacity of the IO-Lix sperm selection protocol to improve sperm parameters (concentration, motility, and sperm DNA fragmentation) of the neat ejaculate. Once...

This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesteron...

Koala retrovirus is a recently endogenized retrovirus associated with the onset of neoplasia and infectious disease in koalas. There are currently twelve described KoRV subtypes (KoRV-A to I, K–M), most of which were identified through recently implemented deep sequencing methods which reveal an animals’ overall KoRV profile. This approach has prim...

Infection with Chlamydia pecorum is one of the main causes of progressive decline of koala (Phascolarctos cinereus) populations in Eastern Australia. Pathological changes associated with the chlamydial infection in the genital tract of female and male koalas have been widely described with reports of acute and chronic lymphoplasmacytic inflammation...

The objectives of this study were to develop a fecal marking protocol to distinguish male from female samples during the echidna breeding season and to determine if normalizing fecal progesterone metabolite data for inorganic content improves the detection of biologically relevant changes in metabolite concentrations. Over a period of 6 weeks, four...

Context: Most of our current knowledge regarding echidna reproductive behaviour is based on qualitative measurements; therefore, it is unclear if specific behavioural cues could be utilised in their captive reproductive management. Aims: This study aimed to identify quantitative changes in general and reproductive behaviour of echidna breeding p...

Observations of 17 heifer-calf pairs were collected over the first 5 days postpartum (p.p.) to study nursing and maternal behaviour of beef cattle. Cattle were managed in a 6 ha paddock and heifer–calf interactions were determined from both regular visual observations and video recordings. Of 17 potential calves, one was stillborn and 3 died in the...

The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic-pituitary-adrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an...

Background: Men with spinal cord injury (SCI) show a high proportion of sperm DNA damage in their ejaculate but the underlying pathology remains elusive. Objective: To investigate the relative incidence of single (SSBs) and double-strand DNA breaks (DSBs) and DNase activity in men with SCI. Materials and methods: This study included ejaculates...

Monotremes (echidnas and platypus) possess five X and four or five Y sex chromosomes, respectively, that evolved independently from the sex chromosomes found in therian mammals. While the platypus has obvious venomous spurs in the male, the short-beaked echidna (Tachyglossus aculeatus) lacks easily identifiable sexually dimorphic characteristics, m...

Zoo and wildlife hospital networks are set to become a vital component of Australia’s contemporary efforts to conserve the iconic and imperiled koala (Phascolarctos cinereus). Managed breeding programs held across zoo-based networks typically face high economic costs and can be at risk of adverse genetic effects typical of unavoidably small captive...

Transmission of Chlamydia pecorum infection has generally been assumed to be via the urogenital route and in an attempt to confirm this we investigated an in vitro method of Chlamydia infection using naturally infected koala semen to inoculate a cell line and attempt to estimate C. pecorum infectious load. A total of 57% of 122 koala semen samples...

This chapter explores the phenomenon of sperm DNA fragmentation (SDF) in the ejaculates of men with spinal cord Injury (SCI). SDF occurs when the DNA molecule of the sperm cell is broken or fragmented because of the concurrence of single-or double-stranded DNA breakages. SDF may arise in the testis during spermiogenesis, during sperm maturation, an...

The determination of oocyte quality is crucial for achieving effective syngamy post-sperm injection and embryonic development. Cumulus cells (CCs) have been proposed as biomarkers of oocyte quality because of their close bio-dynamic relationship with the oocyte. To determine the quality of the oocyte, CCs were sampled during oocyte preparation for...

There is growing evidence that Chlamydia pecorum infection of the male koala reproductive tract causes inflammation and pathology of the urogenital tract. Previous studies have revealed that male koalas exhibiting severe clinical signs of urogenital chlamydial disease had an increased incidence of sperm DNA fragmentation and abnormal sperm morpholo...

Sperm DNA fragmentation (SDF) dynamic assays were piloted on 4 fresh ejaculates to examine the possible sperm toxicity of three common antibiotics, ciprofloxacin, doxycycline and ampicillin, incubated at a concentration estimated to be reached in semen in vivo, and 100×, for 24 h. SDF was assessed in terms of single-strand DNA breaks (SSBs) and dou...

In the short-beaked echidna, Tachyglossus aculeatus , after an initial period of in utero development, the egg is laid in the pouch and incubated for 10 days. During this time, fetuses develop an egg tooth and caruncle to help them hatch. However, there are only a few historical references that describe the development of the monotreme egg tooth. U...

Significance A conserved retrovirus, koala retrovirus (KoRV)-A, is present within the genome of koalas in most Australian populations. Additional divergent sequences and subtypes are thought to be exogenously transmissible and more pathogenic. We present a comprehensive analysis of KoRV genetic diversity within two captive koala populations and sta...

Monotremes diverged from therian mammal ancestors approximately 184 million years ago and have a number of novel reproductive characteristics. One in particular is their penile morphology. There are differences between echidna and platypus phalluses, but both are somewhat similar in structure to the reptilian phallus. The echidna penis consists of...

Research Question Previous studies have demonstrated that human follicular fluid has the potential to preserve sperm chromatin structure and reduce the rate of sperm DNA fragmentation. Human follicular fluid also appears to inhibit seminal plasma DNase activity by an unknown mechanism; the aim of this study was to better elucidate this mechanism....

The aims of this study were to investigate the proteome of koala spermatozoa and that of the prostatic bodies with which they interact during ejaculation. For this purpose, spermatozoa and prostatic bodies were fractionated from the semen of four male koalas and analyzed by HPLC MS/MS. This strategy identified 744 sperm and 1,297 prostatic body pro...

This review reports the current status of artificial breeding technology in the Crocodylia and the future requirements for the establishment of AI in the saltwater crocodile. Although there are challenges regarding safe restraint and immobilisation, semen collection of the saltwater crocodile by manual stimulation has proven effective in yielding s...

Background: The current inability to successfully cryopreserve koala spermatozoa limits the capacity to maintain the genetic diversity of current and future koala populations by means of assisted breeding technologies (ABT). Consequently, this research focused on the prolonged chilled (5 °C) preservation of electro-ejaculated koala semen in order t...

This study describes the progesterone profile during pregnancy in sexually mature female captive short-beaked echidnas (Tachyglossus aculeatus aculeatus). Echidnas were monitored daily by video surveillance to confirm key reproductive behaviour. Plasma samples were collected and pouch morphology assessed three times a week. The pouch of the female...

This study describes the chemical lipid composition of the sperm plasma and acrosomal membranes of the saltwater crocodile Crocodylus porosus with the aim of providing new insights into sperm physiology, particularly that associated with their preservation ex vivo. The specific fatty acid composition of the sperm plasma and acrosomal membranes is d...

This study describes the chemical lipid composition of the sperm plasma and acrosomal membranes of the saltwater crocodile Crocodylus porosus with the aim of providing new insights into sperm physiology, particularly that associated with their preservation ex vivo. The specific fatty acid composition of the sperm plasma and acrosomal membranes is d...

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme linea...

Information on the morphology and histology of the male reproductive system of the Crocodylia species is necessary to determine the role of these tissues in the production of functional spermatozoa. Accordingly, in this study we examined the gross morphology and microanatomy of the testis and the male excurrent duct system through which spermatozoa...

Free-ranging koalas generally feed at night; however, captive koalas are usually fed during the day in order to encourage activity for display purposes. We studied the temporal effect of feeding on body temperature of captive koalas in Queensland, to determine whether nocturnal feeding may be beneficial for koalas in warmer climates. Six adult koal...

The objective of this study was to investigate the effect of acute and chronic heat load events on scrotal temperature (ST), body temperature (BT) and bull behaviour, and to examine the interrelationship between these parameters; the underlying hypothesis was that adverse heat treatments delivered in a temperature controlled environment will lead t...

Although it is well established that chlamydial disease renders female koalas infertile, there has been limited research on its effects on male koala fertility, specifically sperm quality. This study determined whether chlamydial infection adversely affects semen quality of naturally infected koalas and spermatozoa recovered from Chlamydia negative...

Conservation efforts to secure the long-term survival of crocodilian species would benefit from the establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic diversity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodile Crocodylus porosus...

The microencapsulation of spermatozoa offers potential benefits for maintaining sperm survival in vitro. The technique has also resulted in the production of offspring in several domestic animal species, but as yet, it has not been successfully applied in human reproductive medicine. This study examined the effect of alginic acid microencapsulation...

Horses are long-day seasonal breeding animals, however, with modern stallion reproductive management it is important for collection of semen during periods that are not part of the traditional breeding season. This study was conducted to examine variation in the seminal characteristics of individual stallions in Avila, Spain during 1 year with a pa...

Retrospective descriptive study. To study the presence of cell-free DNA (cfDNA) and DNase activity in males with spinal cord injury (SCI) with elevated sperm DNA fragmentation. Hospital in Toledo, Spain; University-based Genetics laboratory in Madrid, Spain. Semen was collected from 15 males with spinal cord injury and elevated sperm DNA fragmentat...

Mammals articulate their jaws using a novel joint between the dentary and squamosal bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before the bones of the novel mammalian jaw joint form. These mammals need t...

Mammals articulate their jaws using a novel joint between the dentary and squamosal bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before the bones of the novel mammalian jaw joint form. These mammals need t...

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed sperma...

Mammals articulate their jaws using a novel joint between the dentary and squamosal bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before the bones of the novel mammalian jaw joint form. These mammals need t...

The distribution of the koala (Phascolarctos cinereus) in Queensland is predicted to contract as a result of climate change, driven by the frequency, intensity and duration of heatwaves and drought. However, little is known about the physiological responses of this species to environmental extremes under field conditions. This study aimed to establ...

Background Procedures to detect sperm DNA fragmentation (SDF), like the Sperm Chromatin Dispersion (SCD) test, determine the “global” SDF without discriminating between spermatozoa with single‐strand DNA breaks only (SDF‐SSBs) and those containing double‐strand DNA breaks (SDF‐DSBs). Objectives (i) To validate a test to distinguish human spermatoz...

Organisms belonging to the Family Chlamydiaceae are responsible for a broad range of diseases in humans, livestock, companion animals and non-domestic species. Infection of the reproductive organs can cause a range of syndromes of which sub- and infertility are the most frequently observed clinical manifestations. While the gross and histological l...

Mammals articulate their jaws using a novel joint between the dentary and squamosal bones. In eutherian mammals, this joint forms in the embryo, supporting feeding and vocalisation from birth. In contrast, marsupials and monotremes exhibit extreme altriciality and are born before the bones of the novel mammalian jaw joint form. These mammals need t...

Retrospective descriptive study. To determine the incidence and probable etiology of sperm DNA fragmentation (SDF) in a sample of males with spinal cord injury (SCI). Hospital in Toledo, Spain; University-based Genetics laboratory in Madrid, Spain. Semen collected by vibro-stimulation from 27 males with various levels of spinal cord injury. Classic...

Purpose To examine the effect of co-incubating spermatozoa with human follicular fluid (HFF) on the rate of sperm DNA fragmentation. Methods This prospective study used semen (n = 23) and HFF from oocyte donors (n = 23). Liquified semen was divided into four aliquots: (1) neat semen (NEAT), (2) seminal plasma removed and replaced with sperm media...

Dual-energy X-ray absorptiometry (DXA) is a non-invasive technology for measurement of body composition that requires validation against reference methods when applied to a new species. The aim of this work was to validate DXA for the assessment of body composition of the echidna. Body composition was determined in the short-beaked echidna (Tachygl...

Background: Within-species skull shape variation of marsupial mammals is widely considered low and strongly size-dependent (allometric), possibly due to developmental constraints arising from the altricial birth of marsupials. However, species whose skulls are impacted by strong muscular stresses - particularly those produced through mastication o...

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a c...

Infectious disease, predominately chlamydiosis, contributes significantly to the decline in health of wild koala (Phascolarctos cinereus) populations in some regions of Australia. In this study, we describe the development and evaluation of a simple, sensitive, and specific loop‐mediated isothermal amplification (LAMP) assay for the detection of Ch...

The successful development and application of an assisted breeding program in any animal relies primarily on a thorough understanding of the fundamental reproductive biology (anatomy, physiology and behaviour) of the species in question. Surely, the ultimate goal and greatest hallmark of such a program is the efficacious establishment of a series o...

'Declining population sizes of koalas (Phascolarctos cinereus) in SE Queensland (QLD), Australia can partially be attributed to chlamydiosis, with the majority of epidemiological studies focusing on the prevalence of infection and associated pathology in female koalas, with lesser attention given to males. We aimed to explore the epidemiology of Ch...

To investigate differences in the post-thaw DNA stability of koala and wombat spermatozoa, protamine amino acid sequences were compared and it was found that there were three more arginine residues for the wombat. Koala and wombat spermatozoa, cryopreserved using identical protocols, were examined for changes in sperm DNA fragmentation (SDF) dynami...

The development of analytical methods for the evaluation of crocodilian semen is an important component for the assessment of male breeding soundness and the development of assisted breeding technology in this taxon. Computer-Assisted Semen Analysis (CASA) technology is becoming an increasingly common technique in seminal evaluations for animals bu...